The worldwide market for proteins produced from recombinant bacteria covers many business sectors beyond biotechnology and medine. The market for enzymes produced by strains of Bacillus alone is estimated to be greater than $1 billion, especially for high-level production of cellulases used to produce biofuels or for other industrial processes. The system is used for high level expression of commercially important proteins; constructing and screening libraries of genes; and complementing bacterial chromosomal mutations. In addition, many strains of Bacillus are sold by the ton commercially for agricultural use because of the their ability to produce secondary metabolites that simultaneously promote plant growth and suppress disease. However, nearly all of these commercially important strains cannot be easily manipulated genetically.
Expression in Bacillus has generally been achieved through gene integration into the bacterial chromosome at a specific site. The drawback of this is that there is only one copy per cell, and expression is not very high. Also, it is difficult to retrieve the gene from the chromosome for additional manipulations. To avoid these issues, several different types of B. subtilis plasmids that have been used, such as pUB110, pE194, pMTLBS72, or pSMbeta1. However, these plasmids are unstable and do not segregate well during cell growth, making them relatively difficult to use for gene expression. During large scale fermentation without antibiotic selection, a significant number of cells (50-99.9%) lose the plasmids. Even under selection, the bacteria may lose their plasmids unless they have this stable segregation system.
Actin, one of the most abundant proteins in the eukaryotic cell, has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria.
Actin is present in all eukaryotic cells and is the most abundant protein of the eukaryotic cytoskeleton. Actin participates in such fundamental processes as cell motility, endocytosis, cell remodeling, cytokinesis, and transcription (Le Clainche et al., Physiol Rev 88:489-513 (2008); Pollard et al., Cell 112:453-465 (2003); Girao et al., FEBS Lett 582:2112-2119 (2008); Wanner et al., J Cell Sci 120:2641-2651 (2007); Pollard Biochem Soc Trans 36:425-430 (2008); Chen et al., Curr Opin Cell Biol 19:326-330 (2007)). Actin is extremely well conserved. The cytoskeletal actins of chicken, cow, and man are identical to each other across all 375 amino acids of the protein. The actin of Saccharomyces cerevisiae is exactly the same length, and its sequence is 89% identical to this vertebrate sequence.
This level of sequence conservation is not required for the actin fold. Actin is a member of a large superfamily of proteins that share the same fundamental architecture. In this superfamily are the 70-kDa heat shock proteins and a group of sugar and sugar alcohol kinases that includes hexokinase and glycerol kinase (Kabsch et al., FASEB J 9:167-174 (1995); Flaherty et al., Proc Natl Acad Sci USA 88:5041-5045 (1991); Bork et al., Proc Natl Acad Sci USA 89:7290-7294 (1992)). The actin folds of rabbit skeletal muscle actin and the 70-kDa heat shock protein from cow, two members of this superfamily, are only 16% identical at the amino acid sequence level, but can be superimposed with a root mean square deviation of 2.3 Å (Flaherty et al., Proc Natl Acad Sci USA 88:5041-5045 (1991)).
Long assumed to lack a cytoskeleton or cytoskeletal proteins, bacteria have in the last decade been shown to contain homologs of actin and also of tubulin and intermediate filaments (Pogliano Curr Opin Cell Biol 20:19-27 (2008); Graumann Annu Rev Microbiol 61:589-618 (2007). To date five distinct families of actin-like proteins have been identified in bacteria, and they are no more related to each other than they are to actin (<13% sequence identity). The crystal structures of members of three of these families, of FtsA, MreB, and ParM, confirmed that their classification as members of the actin family was appropriate despite the very slight resemblance of their sequences to that of actin (van den Ent et al., EMBO J. 19(20):5300-5307 (2000); van den Ent et al., Nature 413:39-44 (2001); van den Ent et al., EMBO J21:6935-6943 (2002)).
MreB is found in many non-spherical bacteria and is required for the generation of proper cell shape (Daniel et al., Cell 113:767-776 (2003); Carballido-López et al., Curr Opin Microbiol 10:611-616 (2007); Osborn et al. Curr Opin Microbiol 10:606-610 (2007)). In Bacillus subtilis, Escherichia coli, and Caulobacter crescentus, helical filaments of MreB coil through the length of the cell at the cytoplasmic membrane (Jones et al., Cell 104:913-922 (2001); Shih et al., Proc Natl Acad Sci USA 100:7865-7870 (2003); Gitai et al., Proc Natl Acad Sci 101:8643-8648 (2004); Figge et al., Mol Microbiol 51:1321-1332 (2004)). The filaments are dynamic, moving in a treadmilling-like fashion (Soufo et al., EMBO Reps 5:789-794 (2004); Kim et al., Proc Natl Acad USA 103:10929-10934 (2006)). FtsA is a component of the bacterial cell division machinery that interacts directly with the machinery's principal component, the tubulin relative FtsZ (Shiomi et al., Mol Microbiol 66:1396-1415 (2007); Pichoff et al., Mol Microbiol 55:1722-1734 (2005)). MamK is present in magnetotactic bacteria and is required for organization into linear chains of the cytoplasmic membrane invaginations that contain magnetic nanocrystals. MamK is assembled into several filaments that flank these chains. In the absence of MamK, the invaginations are disordered and scattered (Komeili et al., Science 311:242-245 (2006); Schüler FEMS Microbiol Rev 32:654-672 (2008)).
ParM and AlfA are each nucleotide-binding components of plasmid partitioning systems. Both form dynamic filaments within the cell, and the dynamic properties of the filaments are required for partitioning (Møller-Jensen et al., EMBO J 21:3119-3127 (2002); Møller-Jensen et al., Mol Cell 12:1477-1487 (2003); Campbell et al., J Cell Biol 179:1059-1066 (2007); Becker et al., EMBO J 25:5919-5931 (2006)). The purified ParM is able to polymerize spontaneously in the presence of ATP into filaments that display dynamic instability (Garner et al., Science 306:1021-1025 (2004); Garner et al., Science 315:1270-1274 (2007)). Plasmids are found at the end of ParM filaments both within the cell and in in vitro reconstructions of the system, which is consistent with a mechanism in which plasmids are pushed towards the cell poles (Gerdes et al., Cell 116:359-366 (2004); Møller-Jensen et al., EMBO J 21:3119-3127 (2002); Møller-Jensen et al., Mol Cell 12:1477-1487 (2003); Campbell et al., J Cell Biol 179:1059-1066 (2007); Garner et al., Science 315:1270-1274 (2007); Garner et al., Science 306:1021-1025 (2004); Salje et al., Science 323:509-512 (2009)). Reconstructions from cryo-electron microscopy indicate that ParM filaments and actin filaments are constructed very differently. The monomer interfaces are different, and as a consequence, ParM and actin filaments are of the opposite helical handedness (Orlova et al., Nat Struct Mol Biol 14:921-926 (2007); Popp et al., EMBO J 27:570-579 (2008)).
With a mere five families of distant relatives identified, actin would appear to have only very sparse representation in bacteria. There are in contrast a great number of actin relatives that have been identified in eukaryotes, and even among these eukaryotic proteins there is considerable sequence and functional diversity. The actin-related proteins, or ARPs were discovered about twenty years ago. Although there exist structures for only Arp2 and Arp3, the secondary structural elements of the actin fold appear to be present in all of the ARPs (Muller et al., Mol Biol Cell 16:5736-5748 (2005)). Arp1, a component of the dynein activator complex, is the closest to actin in amino acid sequence; the sequences of Saccharomyces cerevisiae Arp1 and actin are 46% identical. Arp1 retains the signature property of actin: Arp1 polymerizes into filaments with the pitch of filamentous actin. Arp1 also binds ATP, and filament formation, as in actin, is accompanied by ATP hydrolysis. There are, however, differences. Kinetic profiles indicate that there is no barrier to nucleation and that the Arp1 filaments cannot be extended beyond a specific length (Bingham et al., Curr Biol 9:223-226 (1999)). The divergence is greater for Arp2 and Arp3, which in Saccharomyces are respectively 39% and 32% identical to actin. Their crystal structures, which were solved in the context of the bovine Arp2/3 complex, revealed that the actin fold is well preserved in both proteins (Robinson et al., Science 294:1679-1684 (2001); Nolen et al., Proc Natl Acad Sci USA 101:15627-15632 (2004)). But neither protein homopolymerizes into filaments, each binds ATP with three orders of magnitude lower affinity than actin does, and Arp3 does not appear to hydrolyze ATP at all (Dayel et al., Proc Natl Acad Sci USA 98:14871-14876 (2001); Dayel et al., PLoS Biol 2:0476-0485 (2004)). The remaining ARPs diverge still further from actin. The sequences of Saccharomyces Arp9 and actin, for example, share only 14% identity, on the order of the bacterial actins.
A recent survey of a single eukaryotic genome, Dictyostelium discoideum, turned up 16 genes that code for proteins that closely resemble actin, as well as eight ARPs, in addition to 17 copies of the actin gene, (Joseph et al., PLoS ONE 3:e2654 (2008)).
Genetic competence is the ability of a bacterial cell to take up exogenous DNA and is key to the genetic manipulation of bacteria. In a few strains of Bacillus, such as B. subtilis strain 168, genetic competence can be induced easily, and comes about when the com genes, which encode the DNA uptake machinery are expressed during stationary phase by the transcription factor ComK. In contrast to strain 168, the vast majority of Bacillus strains of commercial importance cannot be readily made competent despite the fact that they contain the same com genes. The inability to activate competence severely limits the ability to manipulate these strains genetically.
In the commonly used laboratory strain Bacillus subtilis 168, competence requires the expression of a set of com genes whose products assemble into a complex in the inner membrane that actively translocates DNA into the cell. Expression of the com genes is under the control of the transcription factor ComK, and cells become competent when ComK accumulates in the cells. Many strains of Bacillus contain all of the com genes necessary for competence, but do not express them. Expression of the B. subtilis ComK protein in these untransformable strains is sufficient to make them competent, but because these strains are untransformable, it is difficult if not impossible, to introduce a ComK expression plasmid into these strains.
The present invention provides additional bacterial proteins that share structural and functional characteristics with actin. The invention thus provides a number of bacterial Actin-like proteins (ALPs). The ALPs can be used to confer stable segregation of any self-replicating DNA molecule (e.g., a plasmid or other expression vector) through multiple generations.
The invention overcomes the longtime limitations associated with protein expression in bacteria, and offers the ability to manipulate many different species of Bacillus. The ability to genetically manipulate these strains will allow their products to be produced at higher yields with increased safety and at reduced costs. The invention further provides an expression vector that is capable of being delivered directly into strains of Bacillus and activating the competence pathway. This general system will allow many species of Bacillus of industrial importance to be easily manipulated genetically.
The invention provides for the first time a plasmid vector that is stably inherited in Bacillus bacterial strains in the absence of antibiotic selection. The vector can be used without further development to produce heterologous proteins in bacteria. The ALPs can be used in such methods. Further included is a system for genetic competence, i.e., the ability to take up exogenous DNA, that is stably inherited.